外源性分化抑制因子Id2在C2C12细胞中的表达

目的：构建大鼠Id2基因真核荧光表达载体，并观察外源性Id2基因C2C12细胞中的表达。方法：RT-PCR扩增出Id2全长cDNA，T4DNA连接酶将载体pGEM-T和Id2cDNA进行连接，构建克隆载体，经限制性内切酶EcoRI酶切pGEM-Id2克隆载体和pEGFP-C2真核表达载体，构建出重组真核表达载体pEGFP-C2-Id2，经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性；通过电穿孔转染法将外源性Id2基因导入C2C12成肌细胞中。分别于转染4、8、12、24、36、72h后通过荧光倒置显微镜下观察细胞整体情况，并计算转染效率。结果：经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向I（12cDNA片段，获得高转染率和高表达外源性Id2基因的C2C12细胞，转染8h时，转染效率约为（10．5±2．8）％；转染12h后，转染效率约为（20．9±3．1）％；转染24h后，转染效率最高，约为（60．8±3．2）％。结论：成功构建了同时携带有G418筛选位点和Id2基因的真核表达载体；并获得高表达外源性Id2基因的C2C12细胞。

The expression of external Id2 protein gene containing green fluorescence in C2C12 cells

Objective ：To construct the eukaryotic expression vector of rat Id2 and to observe the expression of Id2 in C2C12 cells for further study on skeletal muscle regeneration. Methods： RT-PCR method was used to amplify the entire Id2 eDNA. The pGEM-T and Id2 eDNA were ligated by T4 DNA ligase. The cloning vectors and the pEGFP-C2 （eukaryotic expression vector） were first cut by EeoR I and then ligated with Id2 by T4 DNA ligase again. The enzyme analysis and DNA sequencing were used to confirm the recombined vectors. The pEGFP- C2-Id2 vectors were transferred into C2 C12 cells by electric perforation. Fluorescence inverted microscopy was used to observe the global growth of the cells and to calculate the transfection efficiency 4,8,12,24,36 and 72 hours post-transfection. Results：The enzyme analysis and DNA sequencing analysis confirmed that the right Id2 gene was cloned. The Id2 transferred C2C12 cells with high expression and high transfeetion efficiency were obtained. The transfection effieiencies at 8 and 12 hours were respectively （ 10.5 ± 2.8 ） % and （ 20.9 ± 3. 1 ） % , and reached the peak at 24 hours （60.8 ± 3.2% ）. Conclusion：We have successfully constructed the pEGFP-C2-Id2 eukaryotic expression vectors which contain G418 selected site and Id2 gene. We have also obtained C2C12 cells with high expression of external Id2 gene.